Biological signals are transmitted via molecular communication. While a great deal of attention has been directed at characterizing the structural basis of intermolecular communication in protein complexes, little is known about how signals propagate across distances within individual globular proteins or domains. Such events occur in allosteric proteins, components of molecular recognition, ligand receptors, and enzymes. Site-site communication across distances is therefore a fundamental feature of proteins. Atomic resolution studies of intramolecular communication are needed to explain sequence-based modulations of protein activity, substrate specificity, ligand regulation, and distal mutations conferring drug resistance. The influence of distal residues on a protein's active site will be particularly valuable for protein design. The research in this proposal seeks to determine the mechanistic basis for site-site communication using a combination of biophysical and biochemical approaches. Three specific aims address the central hypothesis for this proposal - that side-chain dynamics mediate long-range communication in proteins. Experiments will be carried out on eglin c, a small serine protease inhibitor possessing favorable properties to aid development of the proposed novel approaches. Detection of long-range, pair-wise communication, or "coupling", will be made from extensive thermodynamic double-mutant cycle analysis. Coupling networks will also be mapped through propagated changes in side-chain dynamics, as observed from NMR H spin relaxation measurements in solution. To test mechanisms of signal propagation and the evolution of communication networks from a design perspective, "on-pathway" mutations will be made in order to modulate communication between sites. The functional significance of communication pathways that connect with the active site inhibitory loop will be assessed from a kinetics-based inhibition assay. The mapping of entire coupling networks through the approach presented is general and should be applicable to other proteins and protein complexes.